RNA interference (RNAi) is a naturally occurring biological process by which double-stranded ribonucleic acid (dsRNA) silences (knocks down) target gene expression in a sequence specific manner. Cellular enzymes use dsRNA to target and cleave single stranded RNA (ssRNA), including messenger RNA (mRNA) and non-coding RNA. RNAi is known to occur in many eukaryotes, including plants, fungi, and animals, and offers great potential for selective and efficient regulation of gene expression.
The dsRNA has an antisense strand containing sequence complementary to a sequence in the mRNA or non-coding RNA and a sense strand substantially identical to the sequence in the mRNA or non-coding RNA. The sense and antisense sequences can be present on separate RNA strands or on a single strand. When present on a single strand, the complementary sequences are connected by a non-hybridizing hairpin or loop sequence.
RNAi-mediated gene suppression on targeted weeds, insects, and fungi affecting crops described in the prior art has been achieved using exogenously supplied unmodified dsRNA (UdsRNA) (U.S. Pat. No. 9,121,022; Ivashuta et al. 2015; US Publication No. 20160215290; Koch et al. 2016). It has been found, that when dsRNAs are used to induce RNAi in insects and are supplied in the insects' diet, 60 base pair (bp) or longer dsRNAs are sometimes required for efficient uptake and processing (Bolognesi et al. 2012).
Preparation of UdsRNA longer than about 30 base pairs (bp) has been achieved by in vitro transcription (Timmons 2006) and by fermentation (Fire et al. 1998). Commercially feasible large-scale methods needed for crop protection applications for preparation and purification of the UdsRNA has been described (US Publication No. 20160177299). However, UdsRNAs are sensitive to degradation by nucleases in the environment and the host, reducing efficacy of inhibition of gene expression (Baum 2016).
DsRNA degradation has been addressed in in vitro and in vivo research and for human therapeutics (Ku et al. 2015) by using chemical synthesis of small (<30 bp) interfering dsRNAs (siRNA) with nucleotides modified by chemical means. Preparation of siRNA with chemically modified nucleotides involves sequential protection-deprotection chemical reactions for each nucleotide added in the elongating single strand RNA (ssRNA) chain (Micura 2002). The complexity and expense of such processes are significantly increased for RNA molecules longer than about 30 bp which trigger RNAi (RNAi triggers). While chemically synthesized siRNAs targeting insects using nucleotides chemically modified at the 2′-OH position of the ribose have been also described (Gong et al. 2013), the cost and synthetic complexity of modified siRNAs is neither economically feasible or sufficiently scalable, for preparation of amounts larger than a few grams, or of chemically modified dsRNA longer than about 30 bp.
Post-transcriptional chemical modification of single strand RNA (ssRNA) has been used for analytical purposes, as described in U.S. Pat. No. 6,867,290. SsRNA chemically modified at the 2′-OH position was described as a template to produce UdsRNA in a subsequent step, thereby providing a means for amplification and subsequent detection of minute amounts of unmodified ssRNA. The ssRNA modification was carried out using dimethylsulfoxide (DMSO) as solvent with water present at 5% or less.
Post-transcriptional chemical modification of ssRNA for analytical purposes was also described by Merino (2005). Merino reacted ssRNA in aqueous media containing 10% DMSO with N-methylisatoic anhydride (NMIA) to produce the 2′-O-esters of N-methyl-anthranilic acid at single stranded nucleotides. Derivatization by this method was inefficient. Less than 15% of ssRNA chains in a reaction vessel were modified and those that were modified had, on average, a single 2′-O-ester of NMIA per ssRNA chain. Under these conditions, dsRNA reacted more than 80 times less efficiently, with less than 0.18% of nucleotides in a stem region being modified and only within one (1) nucleotide of the end of a stem (i.e. within one nucleotide of a single strand region) Similar results have been observed for reaction of RNA with other reactants (Nodin 2015). 1-methyl-7-nitroisatoic anhydride (1M7), benzoyl cyanide (BzCN), 2-methyl-3-furoic acid imidazolide (FAI), and 2-methylnicotinic acid imidazolide (NAI) have been used to post-transcriptionally produce 2′-ribose esters of RNA, but have a similarly low percentage of the modification, with modification primarily occurring at riboses of unpaired nucleotides or their immediately adjacent paired nucleotides.
RNAi provides a promising approach to reducing, managing, or controlling pests and weeds in agricultural and urban settings. However, current RNAi technology, using RNAi triggers consisting of UdsRNA or highly modified synthesized siRNA is cost prohibitive for use in agricultural or urban settings. Further, current siRNA production isn't sufficiently scalable. There remains a need for RNAi triggers for agricultural and urban applications that can be economically produced in large scale, stable enough to resist degradation until the point of use, active enough to elicit an effective pesticidal or herbicidal response, and selective enough be considered safe for mammals, humans in particular, and the environment.